Method of producing 1,4&#39;-dihydroxy-3-n-pentyl-6,6,9-trimethyl-6a,7,10,10a-tetra-hydrodibenzo(b,d)pyran

ABSTRACT

THE COMPOUND 1,4&#39;&#39;- DIHYDROXY -3-N-PENTYL-6,6,9TRIMETHYL - 6A,7,10,10A - TETRAHYDRODIBENZO (B,D)PYRAN, WHICH CAN BE PREPARED BY MICROBIAL TRANSFORMATION OF 1-HYDROXY-3-N-PENTYL - 6,6,9- TRIMETHYL-6A,7,10,10A-TETRAHYDRODIBENZO (B,D)PYRAN. THE COMPOUND IS USEFUL AS AN ANTI-DEPRESSANT AGENT,

s,s22,1ss" 1 METHOD OF PRODUCING 1,4' DIHYDROXY-3-n- PENTYL- 6,6,9TRIMETHYL-6a,7,10,10a-TETRA- "HYDRODIBENZO[b,d]PYRAN I Earl Elmer Fager,Lake Villa, and Norman Earl Wide- 5 burg, Waukegan, Ill., assignors toAbbott Laboratories, .''North Chicago, Ill.

No Drawing. Original application Oct. 19, .97.2,.Se1.'.. No. ,298,900.Divided and this application Ai 1g. j1, 1973, Ser. No. 384,554

' Int. Cl. C07c 67/00" US. Cl. 195-51 R 4' Claims ABSTRACT OF THEbisctosm" The compound 1,4 dihydroxy 3 n pentyl-6,6,9- trimethyl6a,7,10,10a tetrahydrodibenzo[b,dlpyran, which canbe prepared bymicrobial transformation of l-hydroxy-S-tm-pentyl 6,6,9trimethyl-6a,7,10,10a-tetrahydrodibenzo[b,d]pyran. The compoundisvuseful. as an antietlepress ant agent. l

DETAILED DESCRIPTION OF THEINVENTION This is a divisionrof application.Ser. No. 29:8,9001filed Oct. 19,1972.

This invention relates to"1,4' dihydroxy 3 n. pentyl- 6,6,9 -tri1nethyl6a,7,10,10a tetrahydrodibenzo[b,d] pyran and to methods for itspreparation. The derivatiye is produced by microbial transformation ofv1-hydr'oxy-3- n 'pe'n'tyl 6,6,9 trimethyl 6a,7,10,10atetrahydrodibenzo[b,d]pyran with active cultures or resuspended washedcells.

The compound of this. invention represented by formula I The compound isproduced by subjecting 1 hydroxy- 3 in pentyl 6,6,9 trimethyl6a,7,10,10a 'tetrahydrodibenzo [b,d] pyran of formula II to microbialtransformation. using "at suitable micr'o organism. I

1,4 Dihydroxy 3-'"-.-''n pentyl 6,6,9 trimethyl- 6a,7,10,l0atetrahydrodibenzo[b,dlpyran is useful'ias an United States Patent 0 "ice3,822,188 Patented July 2, 1974 substrate (H) is added to thefermentation after a heavy growth of mycelium has been obtained, usuallywithin 48 hours after inoculation The substrate, being a gum, can bedissolved in high concentration in acetone or other suitable solventsfor tansfer into the fermentation. The prefflid evel of substrate in thefermentation is between 0.05 and 1.0 g./lit'er{ Incubation and agitationare con tinued after addition of the substrate as necessary for themicrobiological hydroxylation.

Progress of the microbiological hydroxylation can be determined asfollows: A 10 ml. aliquot of the whole culture is removed from thefermentation vessel and mixed with 10 ml. of acetone. Then the sample isextracted with 20 ml. of ethyl acetate. The extract is reduced to aresidue and the residue is extracted with 2 m1. of acetone. A 100 pl.aliquot of the actone solution is applied to a chromatographic plateprecoated with a 0.25 mm. thick layer of silica gel GF-254. Thechromatographic plate is developed with diethylether in a chromatographytank with a saturated atmosphere, open-faced or with a cover plate toform sandwich plate condition. The developed plate is dried of solventand sprayed with a solution of Fast Blue B Salt (0.15% inwater). Thesubstrate and transformation products are visible asdark red spots on awhite background.

Substrate.('lI) is found at R; 0.7-0.85 and the desired product (I) isfound at R; 0.40-0.45.

The fermenation is harvested at the time of maximum yield of the desiredproduct. The desired product may be separated from the culture byextraction of the whole culture at an appropriate pH with a waterimmiscible solvent .such as ethyl acetate or butanol, or with a mixtureof solvents such as ethyl acetate and acetone. Alternatively,

the whole culture may be filtered and the filtrate extracted with thesolvents indicated and the mycelium cake extracted with water misciblesolvents such as actone, methanol or ethanol. The desired product in theresidue, after evaporation of the extraction solvent, can be purified bychromatography.

The following examples further illustrate this invention:

Example 1 Microbial transformation of 1-hydroxy-3-n-pentyl-6,6,9-

trimethyl-6a,7, l 0, IOa-tetrahydro dibenzo [b,d] pyran .Molds,basidiomycetes and actinomycetes, which are :agknown to havehydroxylated other compounds, were selected to test their capacity tohydroxylate 1-hydroxy-3- n pentyl 6,6,9 trimethyl 6a,7,10,10atetrahydrodibenzo[b,d]pyran. The following media were prepared:

Medium I for Molds and Basidiomycetes Grams Soy, Fluff Flour 5 YeastExtract 5 KH PO, v 2.3 zHPOe 1 liter with deionized water, was adjustedto pH 6.5.

Ten ml. aliquots were dispensed into 50 ml. size screw top glass tubeswith stainless steel Morton closures and sterilized by autoclaving at121 C. for 30 minutes. A 50% weightper unit volume glucose solution wasautoclaved separately and added to the sterile ingredients in thefermentation vessel in the amount of 1 m1. of glucose solution to each10 ml. of vessel contents to complete the medium.

Medium II for Actinomycetes Grams Soy Bean Meal 5 Sodium Chloride 1Yeast Extract 2.5 K HPO 2.0 KH2PO4 1.0

in 1 liter with deionizedwater was adjusted to 31 1 6,5-

7.0. Ten ml. aliquots were dispensed into 50 ml. size screw top glasstubes with stainless steel Morton closures and sterilized by autoclavingat 121 C. for 30 minutes. A 50% weight per unit volume glucose solutionwas autoclaved separately and added to the sterile ingredients in thefermentation vessel in the amount of 1 ml. of glucose solution to each10 ml. of vessel contents to complete the medium.

Tubes of complete sterile medium were inoculated in duplicate with oneof the micro-organisms. One seven day old slant of each micro-organismwas suspended as completely as possible in 5 ml. of sterile brothcomposed of 5 grams of glucose, 3 grams of peptone, 3 grams of beefextract, and 1 gram of yeast extract in 1 liter of deionized water andadjusted to pH 7 .0. A 1 ml. portion of the broth suspension was used toinoculate each tube. The inoculated media were incubated at 28 C. on arotary shaker at 250 r.p.m. The cultures showing heavy growth after 48hours incubation, and the remaining few that required 72 hoursincubation, received 5 mg. of substrate (II) in one of the tubes and theduplicate tubes served as a culture control without added substrate. Thesubstrate was added as an acetone solution (50 mg./ml., 0.1 ml. pertube) and all of the cultures were re-incubated on the shaker for anadditional 48 hours. Then each of the cultures was mixed with 10 ml. ofacetone and ml. of ethyl acetate, shaken vigorously to effectextraction, and centrifuged.

.- Ezsam le per flask. The inoculated medium was incubatedat .28

The upper solvent phase from each tube was reduced to a residue and theresidue was extracted with 2 ml. of acetone. Each acetone solution wasapplied at 100 1. to a chromatographic plate precoated with a 0.25 ml.thick layer of silica gel GF-254. The plate was developed with diethylether, dried of solvent and sprayed with Fast Blue B salt reagent.Results are summarized in Table I following. This table lists cultureswhich yielded transformation products with an R of 0.40-0.45. Therelative intensity of the red color produced by the reagent is indicatedas barely visible (1), clearly visible and more intense or The mostintensely visible spot, indi cated by represents a 2-4% conversionof'substrate added to the fermentation. The code following the name ofeach organism designates the following:

NRRL-means the culture is on deposit with and available from theNorthern Utilization Research and Development Division, United StatesDepartmentof Agriculture, Peoria, Ill. 61604.

ATCCrneans the culture is on deposit with and available from theAmerican Type Culture Collection, Rock'- ville, Md. 20852.

QMmeans the culture is on deposit with and available from the MycologyLaboratory, US. ArmyNatick Lab'- oratories, Natick, Mass. 01762. I

Codes other than any of these above three means the organism is from theCulture Collection at Abbott'Laboratories, North Chicago, Ill. 60064.

TABLE I Sub- Yield 0! Medium strata, goduet with Organism used R; 1'.40-45.

AspergillusfonsecaeusNRRL67 I .72 Cunninghamella blakealeeana NR- RL1369. I .78

Ciglmnghamella blakealeeana QM I 7- 5 Cunninghamella elegans ATCC 9245.I .78' Cunninghamclla elegans QM t 6784. .75 Cunninghamellabalnien'ATCC9244. I .75 MucorparaaiticusATCC 6476 I .78 :1: AbsidiacylindrospomNRRL2796-.. I .75 Coprtnusscler0tigenusNRRL3306 I .78Streptomyces cinnamoneous NRRL B-1285. II .83 V Streptomycesviridoilavus NRRL B-1548. .83 Streptomyces cinereoerocatus NRRL 3443. II.83 :l: Actinomycete Sp. ACT-45 II .83

C. on a rotary shaker at 250 r.p.m. Twenty-five mg. of substrate (II) in0.5 ml. acetone was added to each flask after 48 hours incubation. Thecontents of all the fiasks were combined after an additional 24 hoursincubation and the whole culture was extracted with a 4 liter-and a 2liter portion of heptane. and 4 times with 2 liter portions of ethylacetate. Then the culturewas filtered andthe solids fraction wasextracted another 6' timeswith-2 liter portions of acetone, allowingtime for .diffusionof the acetone solubles into the acetone. The greaterportion of the desired product was found in these acetone extractions ofthe mycelium. All extracts containing; significant amounts of thedesired product were combined and the solvents were removed. Thebulky'oily residue, dissolved in heptane, was applied to a short,silica. gel GET- 254 column. Solvent mixtures of increasing polarity (byincreasing ethyl acetate concentration in heptane) were used to elutematerials from the column. The bulk of impurities were eluted withheptane and heptanezethyl acetate (80:20) while the desired product waseluted in the latter portion of heptanezethyl acetate (50:50). Finalpurification of the product was accomplished by preparative thinlayerchromatography on silica gel GF-254 developed with diethyl ether. Thesegment of silica gelcontaining the product was eluted with methanol,the eluate residue was extracted with heptanezchloroform to removeresidual silica gel, the heptanezchloroform was removed and the residuewas dried to yield 52.5 mg. .of the desired pure product, as a gum.

The structure of the product was determined by proton magnetic resonanceand mass spectrometry. High resolution mass spectrometry at 50 e.v. gavea molecular ion at m/e 330.2197 .(calculated for C H O 330.2195 Theprominentfragment ions and their assignments are given in Table II.Comparisonof these data with the mass spectrum of1-hydroxy-3-n-pentyl-6,6,9-trimethyl-6a,7,10,

IOa-tetrahydrodibenzo[b,d]pyran [H. Budzikiewicz et al., Tetrahedron,21, 1881 (1965); U. 'Claussen et al., Tetrahedron, 22, 3535 (1966)]showed that the product was a monohydroxylated derivative ofl-hydroxy-B-npentyl-6,6,9-trimethyl 6a,7,10,10 tetrahydrodibenzo[b,d]pyran. The peak at m/e 258 suggests that the position ofhydroxylation is on the n-pentyl side chain. The exact position ofhydroxylation was determined by proton magnetic resonance spectrometry.The spectrumwas determined at MHz. in CDC]; with tetramethylsilane asthe internal'standard. The chemical shifts are given in Table III. Theassignments were made by. analogy. with the spectrum of1-hydroxy-3-n-pentyl-6,6,9 trimethylrfia,7,1-0,l0a-tetrahydrod@enzo[b,d]pyran ['RwArArcher et al., J. Amer. Chem.S0c.,'92, 5200' (1970)].' The only resonances present in the spectrum ofthe product and not present in the spectrum of 1-hydroxy-3- n -pentyl-6,6,9-trimethyl 6a,7,l0,10a 'tetrahydrodibenzolbl] pyran are the sextetat 3.82 ppm. and the doublet at 1.18 p.p.m. These can arise only fromthe hydroxylation o f the product at the 4'-position. V 1

TABLE II Mass spectrum of1,4-dihydroxy-3-n-pentyl-6,6,9-trhnethy1-6a,7,10,10a-

tetrahydrodibenzo[b,d]pyran Relative intensity Assignment TABLE IIIProton magnetic resonance spectrum of 1,4-dihydr0xy-3-n-pentyl-6,6,9-trimethy1 6a,7,l0,l0a tetrahydrodibenzo[b,d]pyran 1 Thechemical shifts are given in parts per million. Abbreviations: s:singlet, d=doublet, t=triplet, m=multiplet, ddzdoublet of doublets,br=broad.

The compounds useful in the practice of this invention can be formulatedinto various pharmaceutically acceptable dosage forms such as tablets,capsules, pills and the like, for immediate or sustained release, bycombining the compound with a suitable pharmaceutically acceptablecarrier or diluent according to methods well known in the art. Suchdosage forms may additionally include lubricants, excipients, binders,fillers, flavoring and sweetening agents and other therapeutically inertingredients necessary for the formulation of the desired preparation.

We claim:

1. A method of producing 1,4'-dihydroxy-S-n-pentyl- 6,6,9-trimethyl6a,7,l0,10a tetrahydrodibenzo[b,d] pyran by microbial transformation of1-hydroxy-3-npentyl-6,6,9-trimethyl 6a,7,10,10a tetrahydrodibenzo[b,d]pyran comprising the steps of inoculating a microorganism selectedfrom the group consisting of Cunninghamella blakesleeana NRRL 1369,Aspel'gillus fonsecaeus NRRL 67, Cunninghamella blakesleeana QM 631,Cunninghamella elegans ATCC 9245, Cunninghamella elegans QM 6784,Cunninghamella baim'eri ATCC 9244, Absidia cylindrospora NRRL 2796,Coprinus sclerotigenus NRRL 3306, Streptomyces cinnamoneous NRRL B-l285,Streptomyces viridoflavus NRRL B-l548, or Actinomycete ACT-45 into afermentation medium and incubating for a sufiicient time to allowgrowth; adding said l-hydroxy-3-n-pentyl 6,6,9trimethyl-6a,7,l0,l0atetrahydrodibenzo[b,d]pyran substrate to thefermentation medium, incubating said inoculated medium containing saidsubstrate for a time sufficient to allow the conversion to take place;and isolating said l,4'-dihydroxy- 3-n-pentyl-6,6,9-trimethyl6a,7,10,10a tetrahydrodibenzo [b,d] pyran.

2. The method of claim 1 wherein the microorganism is Cunninghamellablakesleeana NRRL 1369.

3. The method of claim 2. wherein said microorganism is incubated for 24to 72 hours prior to the addition of said substrate.

4. The method of claim 3 wherein said substrate is added to saidinoculated fermentation medium in an amount of from 0.05 to 1.0 g./literof medium.

References Cited UNITED STATES PATENTS 3,388,136 6/1968 Taylor et al.260-3453 OTHER REFERENCES Archer et al., JACS 92, pp. 5200-6 (1970).

Chem. Abstracts 74: 1376s (1971).

Allen et al., J. of Bacteriology, August (1970), pp. 426-34.

A. LOUIS MONACELL, Primary Examiner T. G. WISEMAN, Assistant ExaminerUNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Peteht No.3,822,188 Dated July 2, 1974 Invent r( Earl Elmer Fager, Norman EarlWideburg It is certified that error appears in the above-identifiedpatentand that said Letters Patent are hereby corrected as shown below:

Under Column 5, Table-II, delete the line that reads '258 I 23 M-C H andsubstitute therefor:

"258 23 M-C H 0-- Sigped'and sealed this 11th day of February 1975.

(SEAL) Attest:

C. MARSHALL DANN RUTH c. MASON Commissioner of Patents Attesting Officerand Trademarks 7

